Dual-label immunohistochemical study of interleukin-4-and interferon-gamma-expressing cells within the pancreas of the NOD mouse during disease acceleration with cyclophosphamide

Beta cell destruction has been shown to occur when rodent or human islets a re exposed in vitro to inflammatory cytokines, such as interleukin-1 beta ( IL-1 beta), tumour necrosis factor-ct. (TNF-alpha) and interferon-gamma (IF N-gamma). Other cytokines such as interleukin-4 (IL-4) or interleukin-10 (I L-10), when given to NOD mice, prevent insulin-dependent diabetes mellitus (IDDM). In this study, we have employed immunofluorescence histochemistry t o study the expression of IFN-gamma and IL-4 in the pancreas of female NOD mice at various time-points (days 0, 4, 7, 11 and at onset of diabetes) fol lowing disease acceleration with cyclophosphamide (Cy). Dual-label confocal and light microscopy were employed to determine the precise cellular sourc es of the two cytokines. IL-4 immunolabelling was observed in a few immune cells at days 0, 4, and 7 within the pancreatic islets but in larger number s at day Il and at onset of diabetes. The cytokine was co-localized predomi nantly in CD4 cells, while only a small minority of CD8 cells and macrophag es also expressed IL-4. At days 0, 4, 7 and 11, weak to moderate immunolabe lling for IL-4 was also observed in beta cells. In contrast, immunolabellin g for IFN-gamma within the islets was not observed until day 11 and this la belling persisted at onset of diabetes. It was immunolocalized in macrophag es and to a lesser extent in CD4 cells. Only a few CD8 cells were immunopos itive for IFN-gamma. At day 11, a proportion of beta cells showed weak immu nolabelling for IFN-gamma. During the study period, immunolabelling for IFN -gamma was also observed in a proportion of endothelial cells located in th e intra-islet and exocrine regions of Cy and diluent-treated mice. From day 11 onwards, both the cytokines were observed in some of the peri-vascular regions. Our results demonstrate that during Cy-induced diabetes, there is increasing expression of both IL-4 and IFN-gamma in specific immune cells w ithin the inflamed islets in the late prediabetic stage and at onset of dia betes. Further studies are required to correlate our protein immunohistoche mical findings with in situ cytokine gene expression and to determine wheth er there is a clear Th1 cytokine protein bias at clinical onset of diabetes and immediately preceding it.